Conjugate compounds for preventing and / or treating HBV and / or HDV infection, liver disease, and for targeting NTCP

Abstract

The present invention relates to conjugated compounds comprising a peptide moiety (a), which is preferably a hydrophobically modified preS-derived peptide of Hepatitis B virus or a respective cyclic peptide, and an NTCP substrate moiety (b), which is preferably a bile acid. The present invention further relates to pharmaceutical compositions comprising at least one conjugated compound. The present invention further relates to medical uses of the conjugated compounds and pharmaceutical compositions, such as in the diagnosis, prevention, and / or treatment of liver diseases or conditions, and / or in inhibiting HBV and / or HDV infection. The present invention further relates to methods for the diagnosis, prevention, and / or treatment of said diseases and / or infections.

Description

[Technical Field]

[0001] The present invention relates to conjugated compounds comprising a peptide moiety (a), which is preferably a hydrophobically modified preS-derived peptide of Hepatitis B virus or a respective cyclic peptide, and an NTCP substrate moiety (b), which is preferably a bile acid. The present invention further relates to pharmaceutical compositions comprising at least one conjugated compound. The present invention further relates to medical uses of the conjugated compounds and pharmaceutical compositions, such as in the diagnosis, prevention, and / or treatment of liver diseases or conditions, and / or in inhibiting HBV and / or HDV infection. The present invention further relates to methods for the diagnosis, prevention, and / or treatment of said diseases and / or infections. [Background technology]

[0002] Today, approximately 2 billion people carry serological markers for HBV. Of these, approximately 260 million are chronically infected with HBV. According to the Centers for Disease Control (CDC), 15–25% of chronically HBV-infected individuals are likely to develop hepatocellular carcinoma (HCC) within 10 years if not treated appropriately (Shephard et al., 2006). HBV-associated HCC has a poor prognosis, and HBV has therefore been classified by the World Health Organization (WHO) as the most important naturally occurring human carcinogen. Despite the existence of a preventive vaccine, the number of infections is unlikely to decline in the near future due to the lack of a cure, a growing global population, and limited prevention methods in poor countries.

[0003] HBV is transmitted primarily via the parenteral route. 90–95% of infected, immunocompetent individuals clear the virus, thereby conferring lifelong immune protection. Approximately 5–10% of infected individuals develop chronic hepatitis B (300,000–500,000 in Germany). In contrast, in highly endemic regions, particularly in Central Africa and East Asia, the primary mode of transmission is perinatal mother-to-child transmission. Unfortunately, infection in immunocompetent children leads to a chronic disease course in 90–98% of cases. Therefore, hepatitis B-associated HCC is the most common malignancy in many of these countries.

[0004] Chronic hepatitis delta virus (HDV) infection represents the most severe form of viral hepatitis, resulting in increased rates of cirrhosis and hepatocellular carcinoma compared with HBV monoinfection. HDV is a satellite virus of hepatitis B virus (HBV), meaning that chronically HDV-infected patients are always co-infected with HBV. Currently, an estimated 15 to 25 million people are co-infected with HBV / HDV. However, these numbers remain uncertain due to a lack of reliable epidemiological data. Currently, there are no approved drugs for treating HDV-infected patients. The entry inhibitor, milcurdex B / brevirtide, has shown promising results in phase II clinical trials (Mentha et al., 2019).

[0005] Currently approved optimal therapeutic regimens for the treatment of chronic hepatitis B virus (HBV) infection either address the viral genome replication step after an established infection (lamivudine, adefovir, entecavir, tenofovir) or act as immune system modulators (interferon-α). Unfortunately, only 10–25% of patients maintain a sustained virologic response during such treatment. Therefore, it is of paramount importance to develop novel therapies that target replication steps (e.g., viral entry) at pre-infection stages, which could aid in viral clearance and cure of the infection.

[0006] Despite the availability of prophylactic vaccines and reverse transcriptase (RT) inhibitors, the number of HBV infections and HBV-related deaths worldwide (currently approximately 350,000 per year) is increasing. Approximately two-thirds of primary liver cancers are attributable to persistent HBV infection (Chan & Sung, 2006).

[0007] Specific inhibition of viral entry is an attractive therapeutic concept for controlling and ultimately eliminating acute and chronic infections caused by different viruses. In HBV / HDV co-infected patients, entry inhibition has shown promising results in a subset of patients when administered in combination with interferon-α (Myr 203 trial, ClinicalTrials.gov Identifier: NCT03852719, Wedemeyer et al., 2018; Wedemeyer et al., 2019) and as monotherapy (Myr 202 trial, Wedemeyer et al., 2017; Wedemeyer et al., 2018).

[0008] Human hepatitis B virus (HBV) is a member of the hepadnaviridae family. Hepadnaviruses are the smallest enveloped DNA viruses that replicate their DNA genome via reverse transcription of a pgRNA intermediate. During assembly, the nucleocapsid acquires three viral envelope proteins, named large (L), middle (M), and small (S). They are encoded in a single open reading frame and share an S domain required for membrane anchoring. In addition to the S domain, M contains a 55-amino acid N-terminal hydrophilic extension (preS2), while L is further extended by 107, 117, or 118 amino acids (genotype-dependent), termed preS1 (Urban et al., 2014). Hepatitis D virus (HDV) is a satellite virus that utilizes HBV envelope proteins for hepatocyte entry. The myristoylated preS1 domain of L plays a crucial role in HBV and HDV infectivity through specific interaction with the hepatocyte-specific receptor, sodium taurocholate co-transporting polypeptide (NTCP) (Lempp & Urban, 2017).

[0009] We previously identified lipopeptides derived from the L protein of HBV that block HBV and HDV infection of PHH and HepaRG cells (Gripon et al., 2005; Schulze et al., 2010; e.g., WO2009 / 092611 A1, US10,323,068). These lipopeptides are derived from the N-terminal 47 amino acids of the preS1 domain of HBV genotype D (HBVpreS / 2-48). myr ), containing naturally occurring modifications with myristic acid. The main compound is myrculdex B (see, e.g., Bogomolov et al., 2016; Blank et al., 2016; Wedemeyer et al., 2018).

[0010] In WO2009 / 092612 and WO2012 / 107579, the contents of which are incorporated herein by reference in their entirety, the inventors describe hydrophobically modified preS-derived peptides of HBV and their use as vehicles for the specific delivery of compounds to the liver.

[0011] The present inventors have further identified the receptor responsible for binding of these HBV L protein-derived lipopeptides, namely, sodium taurocholate cotransporting polypeptide (NTCP / SLC10A1) (WO2014 / 072526, WO2014 / 072524, and WO2015 / 014830). See also Ni et al. (2014) and Yan et al. (2012). In WO2017 / 102906 A1, the entire contents of which are incorporated herein by reference, the present inventors describe cyclic NTCP-targeting peptides, such as the cyclic form of myrcudex B, and their use as entry inhibitors.

[0012] It is therefore an object of the present invention to provide improved means and methods for the diagnosis, prevention and / or treatment of liver diseases, such as liver diseases related to NTCP-mediated transport.

[0013] The present invention further aims to improve the methods and means for inhibiting, preventing and / or treating HBV infection and other HBV-related diseases as existent in the prior art, and therefore it is an object of the present invention to provide improved methods and means that allow for targeted and effective inhibition, prevention and / or treatment of HBV infection and related diseases.

[0014] It is a further object of the present invention to provide improved means and methods for the inhibition, prevention and / or treatment of HDV infection and HDV-associated diseases.

[0015] The present invention aims to provide improved means and methods for the inhibition of NTCP as an HBV and HDV receptor and NTCP-mediated transport of natural substrates and further compounds. Summary of the Invention

[0016] According to the invention, this problem is solved by: (a) a peptide moiety, and (b) The NTCP substrate portion corresponding to the bile acid binding site of sodium taurocholate cotransporting polypeptide (NTCP). wherein (a) and (b) are covalently bonded to each other, preferably via a linker or amino acid side chain.

[0017] According to the invention, this problem is solved by: (i) at least one conjugate compound of the invention; (ii) optionally, a pharmaceutically acceptable carrier and / or excipient; The problem is solved by providing a pharmaceutical composition comprising:

[0018] According to the present invention, this problem is solved by providing a conjugate compound according to the invention or a pharmaceutical composition according to the invention for use in medicine.

[0019] According to the present invention, this problem is solved by providing a conjugate compound of the invention or a pharmaceutical composition of the invention for use in the diagnosis, prevention and / or treatment of liver diseases or conditions.

[0020] According to the present invention, this problem is solved by a method for the diagnosis, prevention and / or treatment of liver diseases or conditions, comprising the administration of a therapeutically effective amount of a conjugate compound of the present invention or a pharmaceutical composition of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] Before describing the present invention in more detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For purposes of the present invention, all references cited herein are incorporated by reference in their entirety.

[0022] Concentrations, amounts, and other numerical data may be expressed or displayed in range format. It is understood that such range format is merely for convenience and brevity and should thus be interpreted flexibly to include not only the numerical values ​​explicitly recited as limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. Illustratively, a numerical range of "1 to 21" should be interpreted not only to include the explicitly recited values ​​1 to 21, but also to include single values ​​and subranges within the stated range. Thus, included within this numerical range are single values ​​such as 1, 2, 3, 4, 5...17, 18, 19, 20, 21, etc., and subranges such as 2 to 10, 8 to 15, etc. This same principle applies to ranges reciting only one numerical value, such as "at least 90%." Moreover, such interpretation should apply regardless of the width or characteristics of the range described.

[0023] <Conjugate compounds> As outlined above, the present invention provides conjugate compounds.

[0024] The conjugate compound of the present invention comprises (a) a peptide moiety, and (b) The NTCP substrate portion corresponding to the bile acid binding site of sodium taurocholate cotransporting polypeptide (NTCP). Includes.

[0025] The peptide portion (a) and the NTCP substrate portion (b) are covalently bonded to each other, preferably via a linker or an amino acid side chain.

[0026] <Peptide portion (a)> In a preferred embodiment, the peptide moiety (a) is General formula I H-[(X) m -P-(Y) n ]-R (I) Hydrophobically modified preS-derived peptides of hepatitis B virus (HBV) (In the formula, P is the amino acid sequence NPLGFXaaP (SEQ ID NO: 1), and Xaa is F or L; X is an amino acid sequence having a length of m amino acids, where m is 0 or at least 1; Y is an amino acid sequence having a length of n amino acids, where n is 0 or at least 1; wherein m+n is 5 to 25, preferably 8 to 20, more preferably 8 to 15; H is a hydrophobic modification, located at the N-terminus of P or within X or Y, selected from acylation and addition of hydrophobic moieties R is a C-terminal modification, Preferably, the moiety protects against degradation, selected from amides, D-amino acids, modified amino acids, cyclic amino acids; natural and synthetic polymers such as PEG, glycans, etc. or General formula Ia Cyclo[(X) m -P-(Y) n ] (Ia) Cyclic peptides (In the formula, P, X, Y, m and n are as defined above; carrying at least one hydrophobic modification in the amino acid side chain of X and / or Y; wherein the cyclic peptide is not cyclized within the amino acid sequence of P in SEQ ID NO: 1 and not via an amino acid side chain of P; wherein the hydrophobic modification is acylation or addition of a hydrophobic moiety); or a pharmaceutically acceptable salt thereof is selected from.

[0027] In one embodiment, the hydrophobic modification of peptide moiety (a) is acylation with a C8-C22 fatty acid such as capric acid (C10), lauric acid (C12), myristoyl (C14), palmitoyl (C16) or stearoyl (C18), preferably myristoyl (C14), palmitoyl (C16) or stearoyl (C18), more preferably myristoyl (C14).

[0028] In certain embodiments, the one or more hydrophobic moieties are selected from cholesterol, cholesterol derivatives, phospholipids, glycolipids, glycerol esters, steroids, ceramides, isoprene derivatives.

[0029] Preferably, the peptide moiety (a) is a peptide of general formula I, where m=0-18 and / or n=0-7, preferably m=0-7 and / or n=0-6 (such as m=7 and n=6).

[0030] Preferably, the peptide moiety (a) is a cyclic peptide of general formula Ia, where m=0-18 and / or n=0-7, preferably m=0-7 and / or n=0-6, with the proviso that m+n is at least 1.

[0031] In one embodiment, m+n=0, the peptide has a length of / contains 7 amino acids (i.e., P); m+n=1, the peptide has a length of 8 amino acids / contains 8 amino acids; m+n=2, the peptide has a length of / contains 9 amino acids; m+n=3, the peptide has a length of 10 amino acids / contains 10 amino acids; m+n=4, the peptide has a length of 11 amino acids / contains 11 amino acids; m+n=5, the peptide has a length of 12 amino acids / contains 12 amino acids; m+n=6, the peptide has a length of 13 amino acids / contains 13 amino acids; m+n=7, the peptide has a length of 14 amino acids / contains 14 amino acids; m+n=8, the peptide has a length of 15 amino acids / contains 15 amino acids; m+n=18, the peptide has a length of 25 amino acids / contains 25 amino acids; m+n=25, the peptide has a length of / contains 32 amino acids.

[0032] In a preferred embodiment, the peptide moiety (a) is HBVpreS / 2-21(B)G T NLS VP NPLGFFPDHQLDP SEQ ID NO: 2 HBVpreS / -11-21(B) GGWSSKPRKGMG T NLS VP NPLGFFPDHQLDP SEQ ID NO: 3 HBVpreS / 2-21(D)G Q NLS TS NPLGFFPDHQLDP SEQ ID NO: 4 HBVpreS / -11-21(D) GGWSSKPRKGMG Q NLS TS NPLGFFPDHQLDP SEQ ID NO: 5 HBVpreS / 9-16 NPLGFFPD SEQ ID NO: 6 HBVpreS / 8-16 PNPLGFFPD SEQ ID NO: 7 HBVpreS9-17 NPLGFFPDH SEQ ID NO: 8 HBVpreS8-17 PNPLGFFPDH SEQ ID NO: 9 an amino acid sequence selected from the group Or, each qualification in P: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The amino acid sequence of the present invention may comprise or consist of an amino acid sequence having the following structure:

[0033] In certain embodiments, the peptide portion (a) comprises an additional amino acid for covalently attaching the bile acid moiety, wherein the additional amino acid is an L- or D-amino acid, such as lysine (K), D-lysine (k), D-tyrosine (y), cysteine ​​(C), propargylglycine, azidophenylalanine, azidolysine, azidophenylalanine, homoallylglycine, homopropargylglycine, azidohomoalanine, azidonorleucine, azidophenylalanine, propargyloxyphenylalanine, and acetylphenylalanine, and can be a natural or unnatural amino acid.

[0034] In certain embodiments, the peptide portion (a) is a cyclic peptide comprising an additional amino acid for cyclization, wherein the additional amino acid for cyclization can be a natural or unnatural amino acid such as cysteine ​​(C), allylglycine, propargylglycine, azidophenylalanine, etc.

[0035] <NTCP substrate moiety (b)> Preferably, the NTCP substrate moiety (b) is a natural substrate of sodium taurocholate cotransporting polypeptide (NTCP), preferably a bile acid, bile acid dimer and multimer; or a non-natural substrate of NTCP a drug such as ezetimibe, irbesartan, rosiglitazone, zafirlukast, TRIAC, sulfasalazine, etc.; selected from.

[0036] The non-natural substrate is further described in Donkers et al. (2017).

[0037] Preferably, the NTCP substrate moiety (b) is <t cholic acid (CA: cholate); ursodeoxycholic acid (UDCA: ursodeoxycholate); lithocholic acid (LCA: lithocholate);​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​dimers of ursodeoxycholic acid, such as or bis(5β-cholan-24-oic acid 3β-yl)diethylene glycol (see, e.g., Gouin and Zhu, 1996).

[0040] [ka]

[0041] ; CA-TUDCA

[0042] [ka]

[0043] taurourosodeoxycholic acid (TUDCA)-containing dimers such as;

[0044] CA-UDCA

[0045] [ka]

[0046] mixed dimers of bile acids, such as; and the like.

[0047] <Combination of (a) and (b)> In the conjugate compound, the peptide moiety (a) and the NTCP substrate moiety (b) are covalently linked to each other, preferably via a linker or an amino acid side chain.

[0048] The attachment site of the bile acid moiety is preferably a carboxylic acid group or a sulfonic acid group of the bile acid moiety.

[0049] Attachment is also possible via the ring structure of the bile acid moiety.

[0050] Preferably, the bile acid is not attached to the peptide moiety (a) via the 3-hydroxyl group of ring A of the steroid skeleton.

[0051] The bile acid moiety (b) is preferably attached to the side chain of lysine (K). In this embodiment, the peptide portion may include an additional D-amino acid, such as D-tyrosine (y), to serve as a linker to the lysine.

[0052] Preferred conjugate compounds are: HBVpreS / 2-21-K-myr-CA (genotype B), HBVpreS / 2-21-K-myr-CA (genotype D), HBVpreS / 2-21-K-myr-LCA (genotype B), HBVpreS / 2-21-K-myr-LCA (genotype D), HBVpreS / 2-21-K-myr-UDCA (genotype B), HBVpreS / 2-21-K-myr-UDCA (genotype D), HBVpreS / 2-21-K-myr-(UDCA)2 (genotype B), HBVpreS / 2-21-K-myr-(UDCA)2 (genotype D), HBVpreS / 2-21-yK-myr-CA (genotype B), HBVpreS / 2-21-yK-myr-CA (genotype D), HBVpreS / 2-21-yK-myr-LCA (genotype B), HBVpreS / 2-21-yK-myr-LCA (genotype D), HBVpreS / 2-21-yK-myr-UDCA (genotype B), HBVpreS / 2-21-yK-myr-UDCA (genotype D), HBVpreS / 2-21-yK-myr-(UDCA)2 (genotype B), HBVpreS / 2-21-yK-myr-(UDCA)2 (genotype D), HBVpreS / 2-21-k-myr-CA (genotype B), HBVpreS / 2-21-k-myr-CA (genotype D), HBVpreS / 2-21-k-myr-LCA (genotype B), HBVpreS / 2-21-k-myr-LCA (genotype D), HBVpreS / 2-21-k-myr-UDCA (genotype B), HBVpreS / 2-21-k-myr-UDCA (genotype D), HBVpreS / 2-21-k-myr-(UDCA)2 (genotype B), HBVpreS / 2-21-k-myr-(UDCA)2 (genotype D), More preferably HBVpreS / 2-21-K-myr-CA (genotype B) HBVpreS / 2-21-yK-myr-CA (genotype B) HBVpreS / 2-21-yK-myr-LCA (genotype B) HBVpreS / 2-21-K-myr-UDCA (genotype B) HBVpreS / 2-21-yK-myr-UDCA (genotype B) HBVpreS / 2-21-K-myr-(UDCA)2 (genotype B) HBVpreS / 2-21-yK-myr-(UDCA)2 (genotype B) is.

[0053] [Table 1]

[0054] The bile acid moiety (b) is preferably attached to the side chain of lysine (K). In one embodiment, an amino acid substitution is made to introduce lysine (K) into the amino acid sequence of peptide moiety (a).

[0055] Preferred positions for such amino acid substitutions are: [ka] [ka] [ka] [ka] is.

[0056] Examples of preferred cyclic conjugate compounds are: Myr-cyclo-[HBVpreS2-21(B)]-yK (SEQ ID NO: 2) myr-cyclo C[G T NLS VP NPLGFFPDHQLDPyK]C Myr-cyclo-[HBVpreS2-21(B)]-K (SEQ ID NO: 2) myr-cyclo C[G T NLS VP NPLGFFPDHQLDPK]C Myr-cyclo-[HBVpreS2-21(B)]-k (SEQ ID NO: 2) myr-cyclo C[G T NLS VP NPLGFFPDHQLDPk]C Myr-cyclo-[HBVpreS2-21(D)]-yK (SEQ ID NO: 4) myr-cyclo C[G Q NLS TS NPLGFFPDHQLDPyK]C Myr-cyclo-[HBVpreS2-21(D)]-K (SEQ ID NO: 4) myr-cyclo C[G Q NLS TS NPLGFFPDHQLDPK]C Myr-cyclo-[HBVpreS2-21(D)]-k (SEQ ID NO: 4) myr-cyclo C[G Q NLS TS NPLGFFPDHQLDPk]C is.

[0057] [Table 2]

[0058] Further moieties of the conjugate compound The conjugate compound of the present invention comprises drugs or their respective prodrugs; tag; Labels such as fluorescent dyes, radioisotopes and imaging agents; recombinant viruses; a carrier or depot for a drug, prodrug, or label; immunogenic epitopes; hormone; inhibitors; toxin etc. It may include one or more further moieties.

[0059] The one or more further moieties are preferably linkers, spacers and / or anchor groups, For example, a cleavable linker etc. are covalently bonded.

[0060] The present inventors have discovered that the conjugate compounds of the present invention are bivalent inhibitors of entry inhibition via targeting of sodium taurocholate cotransporting polypeptide (NTCP / SLC10A1). In particular: The peptide portion of the conjugate interacts allosterically with NTCP through its essential domain NPLGFXaaP (SEQ ID NO: 1); Since bile acids are the natural substrates / ligands of NTCP, the NTCP substrate portion of the conjugate interacts with the bile acid site (substrate binding site) of NTCP.

[0061] Through extensive mutational analysis, we have shown that the essential binding site of the peptide (NPLGF(F / L)P) does not directly interact with the bile acid binding site. We have demonstrated that bile acid binding does not preclude interaction with the essential peptidic portion of the new substrate. Notably, blocking both binding sites results in a slower turnover of the receptor, thus extending the half-life of the receptor on the surface of hepatocytes. This, together with the immobilization of the bile salt substrate by the peptide, results in a synergistic effect of both moieties.

[0062] <Pharmaceutical Compositions and Medical Uses> As outlined above, the present invention provides pharmaceutical compositions.

[0063] The pharmaceutical composition of the present invention comprises: (i) at least one conjugate compound of the invention; (ii) optionally, a pharmaceutically acceptable carrier and / or excipient; Includes.

[0064] As mentioned above, the present invention provides a conjugate compound of the present invention or a pharmaceutical composition of the present invention for use in medicine.

[0065] Thus, the conjugate compounds of the invention or the pharmaceutical compositions of the invention are suitable for and therefore provided for the diagnosis, prevention and / or treatment of diseases.

[0066] As mentioned above, the present invention provides a conjugate compound of the present invention or a pharmaceutical composition of the present invention for use in inhibiting HBV and / or HDV infection.

[0067] As mentioned above, the present invention provides the conjugate compound of the present invention or the pharmaceutical composition of the present invention for use in the prevention of primary HBV and / or HDV infection.

[0068] As mentioned above, the present invention provides the conjugate compound of the present invention or the pharmaceutical composition of the present invention for use as an HBV and / or HDV entry inhibitor.

[0069] Preferably, HBV infection of any genotype is inhibited or prevented.

[0070] Preferably, HDV infection is inhibited or prevented for HDV of any genotype, ie, HDV having any type of HBV envelope protein.

[0071] Preferably, the entry inhibition is via targeting of sodium taurocholate cotransporting polypeptide (NTCP / SLC10A1), ie, "NTCP targeting."

[0072] As noted above, in certain embodiments, the conjugate compound further comprises: (i) respond to and inhibit NTCP, particularly as a bivalent inhibitor (as described above); and (ii) targeting to the liver by interacting with NTCP and subsequently interfering with viral replication via the action of a second active domain on the nucleocapsid; It may act as a combination inhibitor.

[0073] As mentioned above, the present invention provides a conjugate compound of the present invention or a pharmaceutical composition of the present invention for use in the diagnosis, prevention and / or treatment of a liver disease or condition.

[0074] In one embodiment, the liver disease or condition is selected from hepatitis, cirrhosis, hemochromatosis, preferably hepatitis caused by hepatitis A, B, C, D, E, F, G and H viruses or concomitant hepatitis caused by multiple viruses.

[0075] In certain embodiments, the liver disease or disorder is a disease involving the liver stage of a viral or non-viral pathogen, such as a tropical disease, malaria, schistosomiasis, leishmaniasis, Wilson's disease, and the like.

[0076] In one embodiment, the liver disease or disorder is a liver tumor, preferably hepatocellular carcinoma (HCC).

[0077] In one embodiment, the liver disease or liver injury is a post-transplant complication following liver transplantation related to bile salt accumulation in the bile pathway / post-transplant related liver dysfunction.

[0078] In certain embodiments, the liver disease or condition involves sodium taurocholate cotransporting polypeptide (NTCP)-mediated transport of a compound into hepatocytes or requires delivery of a drug or labeled compound to the site or location of the disease or condition.

[0079] Preferably, the liver disease or condition is: intrahepatic cholestasis, Liver poisoning (due to liver toxins) / hepatotoxicity, Drug-induced cholestatic liver disease, hyperlipidemia, Posthepatic cholestasis, metabolic syndrome, Non-alcoholic fatty liver disease (NAFLD) glycogen storage disease The liver-related metabolic disease is selected from the group consisting of:

[0080] Preferably, the compounds transported to hepatocytes via NTCP are preferably bile acids, steroids, conjugated and unconjugated thyroid hormones, hepatotoxins, compounds covalently bound to taurocholic acid, bromosulfophthalein, drugs.

[0081] In one embodiment, the conjugate compound of the present invention or the pharmaceutical composition of the present invention comprises immunomodulatory agents, For example, interferon (IFN), interferon lambda, and / or RIG-I, MDA-5 or TLR agonists or antagonists, For example, inarigivir, a TLR-7 agonist and TLR-8 agonist; It is used in combination therapy with another therapeutic agent such as

[0082] Recent in vitro and clinical trials (Myr-203) by researchers have demonstrated strong synergy between IFN and entry inhibitors. In the case of HDV infection, IFN prevents mitosis-mediated cell-to-cell spread of HDV RNA, which cannot be blocked by Myrcurdex B. Thus, administration of both drugs simultaneously demonstrates stronger suppression of viremia. Furthermore, entry inhibition by Myrcurdex has been shown to allow the immune modulator IFN to help reconstitute the adaptive immune system (see Myr-203 study, Wedemeyer et al., 2019; Wedemeyer et al., 2018) and aid in HBV and HDV clearance.

[0083] The present inventors have identified and described sodium taurocholate cotransporting polypeptide (NTCP) as a target and for use in the prevention and / or treatment of certain liver diseases or conditions, such as liver diseases or conditions related to NTCP-mediated transport of compounds (such as bile acids) into hepatocytes, preferably liver-related metabolic diseases (e.g., intrahepatic cholestasis, liver intoxication (due to hepatotoxins) / hepatotoxicity, drug-induced cholestatic liver disease, hyperlipidemia, etc.) and cardiovascular diseases. See WO2014 / 072524 and PCT / EP2014 / 066262, the entire contents of which are incorporated herein by reference.

[0084] "Liver-associated metabolic disease," as used herein, refers to metabolic disorders that are affected by hepatic metabolism of lipids and bile acids, including visceral obesity, diabetes, and dyslipidemia.

[0085] Generally, "cholestasis" is a condition in which bile components cannot be secreted from hepatocytes into the bile ducts or bile cannot flow from the liver to the duodenum, resulting in hepatocellular bile acid accumulation within hepatocytes.

[0086] "Cholestasis" or "intrahepatic cholestasis," as used herein, refers to the intrahepatic toxic effects of hepatocellular bile acid accumulation due to insufficient expression and / or activity of bile salt pumps (such as BSEP or MRP) in the bile canalicular membrane.

[0087] "Posthepatic cholestasis," as used herein, refers to cholestatic liver disease due to obstruction of the large bowel.

[0088] "Liver poisoning" or "hepatotoxicity" or "toxic liver disease," as used herein, refers to the toxic effects of drugs unrelated to bile acid accumulation. These drugs penetrate hepatocytes via NTCP-mediated transport and cause some direct toxic effects by damaging mitochondria or by activating enzymes in the cytochrome P-450 system, resulting in oxidative stress.

[0089] "Drug-induced cholestatic liver disease," as used herein, refers to the inhibition of bile acid export from hepatocytes due to drug effects on the bile salt export pump (BSEP).

[0090] Drug-induced cholestasis can be caused by several drugs that inhibit BSEP, such as rifamycin, cyclosporine A, rifamycin SV, bosentan, troglitazone, erythromycin estolate, and glibenclamide (Fattinger et al., 2001; Funk et al., 2001; Funk et al., 2001; Stieger et al., 2000; Dawson et al., 2012; Morgan et al., 2010; Ogimura et al., 2011). BSEP is a member of the ATP-binding cassette (ABC) family of transporters (also identified as ABCB11) and is involved in the process of exporting bile acids out of hepatocytes, thereby reducing their toxicity to these cells. By preventing BSEP-mediated bile acid export, the aforementioned drugs cause the toxic effects of excessive bile acid accumulation. Inhibition of NTCP-mediated bile acid uptake via lipopeptide-based compounds (such as MyrB) and NTCP counteracts BSEP inhibition, thereby preventing hepatotoxicity or being suitable for treatment and / or diagnosis.

[0091] "Hyperlipidemia" (or hyperlipoproteinemia, or hyperlipidemia) involves abnormally elevated levels of any or all lipids and / or lipoproteins in the blood.

[0092] Hyperlipidemia is divided into primary and secondary subtypes. Primary hyperlipidemia is usually due to genetic causes (e.g., mutations in receptor proteins), while secondary hyperlipidemia results from other underlying causes, such as diabetes. Lipid and lipoprotein abnormalities are common in the general population and are considered modifiable risk factors for cardiovascular disease due to their effect on atherosclerosis.

[0093] "Hypercholesterolemia" (or hypercholesterolemia) is the presence of high levels of cholesterol in the blood. It is a form of "hyperlipidemia."

[0094] "Hyperlipidemia," as used herein, preferably refers to high cholesterol, including elevated LDL cholesterol, decreased HDL cholesterol, elevated triglycerides, clogged arteries leading to high blood pressure, cardiovascular disease (CVD), heart attack, and stroke.

[0095] "Metabolic syndrome" refers to a disorder of energy utilization and storage, diagnosed by the co-occurrence of three of the following five medical conditions: abdominal (central) obesity, elevated blood pressure, elevated fasting blood glucose, high serum triglycerides, and low high-density cholesterol (HDL) levels. Metabolic syndrome increases the risk of developing cardiovascular disease, particularly heart failure, and diabetes. Metabolic syndrome is also known as metabolic syndrome X, cardiometabolic syndrome, syndrome X, insulin resistance syndrome, Lieven syndrome, and CHAOS.

[0096] "Non-alcoholic fatty liver disease" (NAFLD) refers to one cause of fatty liver, which occurs when fat accumulates in the liver (steatosis) without excessive alcohol consumption. It is associated with insulin resistance and metabolic syndrome. Non-alcoholic steatohepatitis (NASH) is the most extreme form of NAFLD and is considered the leading cause of liver cirrhosis of unknown etiology.

[0097] Preferably, NTCP-mediated transport is reduced or blocked by the conjugated compounds of the present invention.

[0098] As noted above, in certain embodiments, the conjugate compound is (i) respond to and inhibit NTCP (as described above), particularly as a bivalent inhibitor; and (ii) targeting to the liver through interaction with NTCP and subsequent delivery of the second active domain to the nucleocapsid to interfere with viral replication; It may act as a combination inhibitor.

[0099] <Route of administration> Preferably, the route of administration of the conjugate compound or pharmaceutical composition of the present invention is selected from oral, subcutaneous, intravenous, nasal, intramuscular, transdermal, inhalation, and by suppository.

[0100] The preferred route of administration or application is oral.

[0101] A preferred embodiment for nasal administration or application is a nasal spray.

[0102] <Therapeutically effective amount> The conjugate compound or pharmaceutical composition of the present invention is provided to contain a therapeutically effective amount of the cyclic peptide or the pharmaceutical composition.

[0103] A "therapeutically effective amount" of a conjugate compound or pharmaceutical composition of the present invention refers to an amount sufficient to inhibit NTCP receptor function. Furthermore, the "therapeutically effective amount" depends on the respective application and the desired result of inhibition, treatment, or vaccination.

[0104] (ii) compared to uses requiring saturation of NTCP (e.g., 1 mg per patient or 1-2 mg / patient), such as for inhibiting NTCP-mediated transport of bile acids, drugs, etc. (i) A different therapeutically effective amount (e.g., 0.01-0.5 mg per patient, preferably 0.1-1 mg / patient) is required for antiviral use or entry inhibition.

[0105] In certain embodiments, the "therapeutically effective amount" of the conjugate compound or pharmaceutical composition of the present invention refers to an amount sufficient to inhibit HBV and / or HDV infection; prevent primary HBV and / or HDV infection; treat hepatitis B and / or D and / or inhibit vaccination and / or entry of HBV and / or HDV in vivo.

[0106] A preferred therapeutically effective amount is in the range of 10 μg to 1 mg, preferably 10 μg to 100 μg per kg of body weight.

[0107] Preferably, a therapeutically effective amount is administered to a patient at a dose ranging from about 0.01 mg to about 50 mg per patient per day, preferably from about 0.1 mg to about 10 mg per patient per day, or from 100 nmol per kg to 2 μmol per kg per day, or from 10 pmol per kg to 20 μmol per kg of body weight.

[0108] IC of the cyclic peptide used, approximately 10 nM 50 For values, a preferred therapeutically effective amount is about 100 μg per kg of body weight, or in the range of 1-5 mg per patient. A preferred therapeutically effective amount in the range of 1-5 mg per patient may be administered once a day, or in other embodiments, only once every 2-3 days.

[0109] A person skilled in the art would be able to determine the appropriate therapeutically effective amount.

[0110] <Methods for diagnosing, preventing and / or treating diseases> As noted above, the present invention provides methods for inhibiting HBV and / or HDV infection and / or preventing primary HBV and / or HDV infection.

[0111] The method comprises administering a therapeutically effective amount of a conjugated compound of the invention or a pharmaceutical composition of the invention.

[0112] Preferably, HBV infection of any genotype is inhibited or prevented.

[0113] Preferably, HDV infection is inhibited or prevented for HDV of any genotype, ie, HDV having any type of HBV envelope protein.

[0114] As outlined above, the present invention provides methods for the diagnosis, prevention and / or treatment of liver diseases or conditions.

[0115] The method comprises administering a therapeutically effective amount of a conjugated compound of the invention or a pharmaceutical composition of the invention.

[0116] In one embodiment, the liver disease or condition is selected from hepatitis, cirrhosis, hemochromatosis, preferably hepatitis caused by hepatitis A, B, C, D, E, F, G and H viruses or simultaneous hepatitis caused by multiple viruses.

[0117] In certain embodiments, the liver disease or disorder is a disease involving the liver stage of a viral or non-viral pathogen, such as a tropical disease, malaria, schistosomiasis, leishmaniasis, Wilson's disease, and the like.

[0118] In one embodiment, the liver disease or disorder is a liver tumor, preferably hepatocellular carcinoma (HCC).

[0119] In certain embodiments, the liver disease or injury is a post-transplant complication after liver transplantation related to bile salt accumulation in the bile pathway.

[0120] In certain embodiments, the liver disease or condition involves sodium taurocholate cotransporting polypeptide (NTCP)-mediated transport of a compound into hepatocytes or requires delivery of a drug or labeled compound to the site or location of the disease or condition; Preferably, intrahepatic cholestasis, Liver poisoning (due to liver toxins) / hepatotoxicity, Drug-induced cholestatic liver disease, hyperlipidemia, Posthepatic cholestasis, metabolic syndrome, Nonalcoholic fatty liver disease (NAFLD) glycogen storage disease a liver-related metabolic disease selected from Here, the compounds transported to hepatocytes via NTCP are preferably bile acids, steroids, conjugated and unconjugated thyroid hormones, hepatotoxins, compounds covalently bound to taurocholic acid, bromosulfophthalein, and drugs.

[0121] In one embodiment, the method comprises: immunomodulators, For example, interferon (IFN), interferon lambda, and / or RIG-I, MDA-5 or TLR agonists or antagonists, For example, inarigivir, a TLR-7 agonist and TLR-8 agonist; This includes combination therapy with another therapeutic agent, such as with other therapeutic agents.

[0122] In the methods of the present invention, and as mentioned above, the "therapeutically effective amount" depends on the respective application and the desired result of inhibition, treatment or vaccination.

[0123] (ii) compared with use requiring saturation of NTCP (e.g., 1 mg / patient or 1–2 mg / patient); (i) A different therapeutically effective amount (e.g., 0.01-0.5 mg per patient, preferably 0.1-1 mg / patient) is required for antiviral use or entry inhibition.

[0124] In certain embodiments, and as noted above, the therapeutically effective amount preferably ranges from about 0.01 mg to about 50 mg per patient, preferably from about 1 mg to about 10 mg per patient; Alternatively, the conjugate compound is administered to the patient at a dose preferably ranging from 10 pmol per kg to 20 μmol per kg of body weight.

[0125] In the methods of the present invention, and as described above, the route of administration is preferably selected from oral, subcutaneous, intravenous, nasal, intramuscular, transdermal, inhalation, and by suppository.

[0126] The following examples and figures illustrate the invention without, however, limiting it thereto. [Brief explanation of the drawings]

[0127] [Figure 1A] Figure 1A shows examples of bile acid (for the bile acid portion (b) of the conjugate compound) monomers showing preferred attachment sites to the peptide moiety. [Figure 1B] Figure IB shows examples of bile acids (for the bile acid portion (b) of the conjugate compound) monomers showing preferred attachment sites to the peptide moiety. [Figure 1C] Figure 1C shows examples of bile acids (for the bile acid portion (b) of the conjugate compound). Bile acid dimers. [Figure 2A] Figure 2A shows a preferred conjugate compound, in which the peptide portion is linear. [Figure 2B] 2B shows a preferred conjugate compound. The peptide portion is a cyclic peptide. Possible positions for substitution at lysine (K) for attachment of the NTCP substrate portion are also shown. [Figure 3]Figure 3 shows an HBV / HDV infection assay. Conjugate compounds containing truncated variants of myrcudex B (amino acids 2-21) conjugated with the bile acids cholic acid (CA) and ursodeoxycholic acid (UDCA) were tested. They were compared with a control peptide lacking the bile acid moiety, i.e., HBVpreS / (-11)-21 myr (genotype B), and the two bile acids CA and UDCA alone. The conjugated compounds exhibit significantly higher activity than the peptides alone (1-2 nM vs. 20-100 nM). [Figure 4] Figure 4 shows an HBV / HDV infection assay. Conjugate compounds containing a truncated variant of myrcudex B (amino acids 2-21) conjugated with cholic acid (CA) and a cyclic peptide (containing amino acids 2-21) conjugated with ursodeoxycholic acid (UDCA) were tested. They were compared with a control peptide lacking the bile acid moiety, i.e., HBVpreS / 2-21myr (genotype B). [Figure 5] Figure 5 shows an HBV / HDV infection assay of compounds conjugated with bile acid monomers and dimers. Three compounds were tested: two conjugated with ursodeoxycholic acid (UDCA) and one conjugated with a dimer of ursodeoxycholic acid (UDCA), where two compounds contain amino acids 2-21 of myrcudex B in a linear form and one compound contains them as a cyclic polypeptide. [Figure 6] 6 shows in vivo planar scintigraphic imaging of an iodine-125 labeled compound of the invention. Time course of scintigraphic imaging (planar imaging) of an 125I labeled compound in naive mice at the indicated time points after injection. [Figure 7]Figure 7 shows the inhibition of infection by bile acid conjugates with linear peptide sequences. Reduction of HDV infection in Huh7-hNTCP cells by various bile acid conjugates, a control peptide, ursodeoxycholic acid, and cholic acid. Cells were pretreated with the substrate for 30 minutes and then co-incubated with the substrate and HDV overnight. Five days after infection, intracellular ELISA was performed (n=3). [Figure 8] Figure 8 shows the inhibition of infection by bile acid conjugates with cyclic peptide sequences. Reduction of HDV infection by various bile acid conjugates and control peptides in Huh7-hNTCP cells. Cells were pretreated with the substrate for 30 minutes and then co-incubated with the substrate and HDV overnight. Five days after infection, intracellular ELISA was performed (n = 3). [Figure 9] Figure 9 shows the inhibition of infection by bile acid dimer peptide conjugates. Reduction of HDV infection by bile acid dimer peptide conjugates compared with monomeric bile acid conjugates and control peptides in Huh7-hNTCP cells. Cells were pretreated with substrate for 30 minutes and then co-incubated with substrate and HDV overnight. Five days after infection, intracellular ELISA was performed (n=3). [Example]

[0128] [Example 1: Solid-phase synthesis of peptide moiety] All peptides were synthesized by automated Fmoc / tBu solid-phase peptide synthesis on an Applied Biosystems 433A synthesizer as previously described (Schieck et al., 2010). Cyclization was achieved under oxidative conditions to form the respective disulfide bridges. After peptide synthesis, myristic acid was attached to the N-terminus of the peptide. To this end, the resin was shaken with a solution of myristic acid (10 equiv.), HBTU (9.5 equiv.), and DIPEA (20 equiv.) in NMP for 2.5 h. The resin was then washed with NMP and DCM.

[0129] For the removal of the lysine side chain protecting group (Aloc), the resin was incubated with a solution of 3 mg of Pd(PPh3) and 30 mg of BH3NHMe2 in DCM for 20 minutes. The resin was washed with DCM and MeOH, and then incubated twice in DCM / MeOH (10:1) for 30 minutes. Subsequently, the resin was washed with DCM and Et2O and dried in vacuo.

[0130] [Example 2: Conjugation of Bile Acid Peptide Conjugates] For the attachment of different bile acids or bile acid dimers, the resin was swollen in NMP. Subsequently, a solution of bile acid (10 equivalents), HBTU (9.5 equivalents) and DIPEA (20 equivalents) in NMP was added and shaken for 2.5 h. Thereafter, the resin was washed with NMP, DCM and Et2O and dried under vacuum.

[0131] The conjugates were cleaved with 95% TFA / 2.5% TIS / 2.5% H2O for 1 h and then precipitated in cold Et2O. Subsequently, they were purified by preparative reverse-phase HPLC, the purity was confirmed by analytical HPLC, and then analyzed by LC-MS.

[0132] [Example 3: HDAg Intracellular ELISA (Huh7-hNTCP)] [A. Materials] Washing buffer: PBS + 0.05% Tween20 Permeabilization buffer: PBS + 0.25% Triton X-100 Blocking buffer: PBS + 0.05% Tween 20 + 1% casein White 96-well cell culture plates: Greiner 655098 Advansta ELISABright chemiluminescent substrate (ordered via Biozym #541025) Hydrogen peroxide solution (35%, Sigma 349887) First antibody Mouse or rabbit anti-HDAg (FD3A7) 1:3000 Second antibody: Goat anti-mouse or goat anti-rabbit conjugated with peroxidase 1:5000

[0133] <B. Procedure> Grow and infect the cells in a white 96-well plate. 1) Fix the cells with 50 μl of 4% PFA for 30 minutes at RT 2) Wash twice with PBS (not the washing buffer) 3) Permeabilize with 100 μl of permeabilization buffer for 30 minutes at RT 4) Block with 100 μl of blocking buffer for 30 minutes at RT 5) Incubate with 100 μl of primary antibody (1:3000) diluted in blocking buffer for 1 hour at RT with shaking 6) Wash with 200 μl three times (washing buffer) 7) Incubate with 100 μl of 3% hydrogen peroxide solution (1:12 from stock in PBS to 3% for Huh7-derived cells) for 10 minutes at RT 8) Wash with 200 μl four times (washing buffer) 9) Incubate with 100 μl of the second antibody at 1:5000 in blocking buffer for 1 hour at RT with shaking 10) Wash with 200 μl three times (washing buffer) + wash with 200 μl two times for 10 minutes each (washing buffer) + wash with 200 μl once for 10 minutes (permeabilization buffer) [Very important] 11) Add 50 μl of luminescence substrate (mix according to the manufacturer's protocol), after pipetting, measure directly with a plate reader. (Enhanced luciferase protocol, measure for 0.1 seconds)

[0134] <B. Results> The results are shown in Figures 3, 4 and 5 (genotype B sequences). The following IC50 values were measured: UDCA: 79 μM CA: 115 μM HBVpreS / 2-21myr: 62.8nM HBVpreS / 2-21myr-CA: 7.27nM HBVpreS / 2-21myr-UDCA: 8.45nM Cyclo2-21-UDCA: 10.1 nM HBVpreS / 2-21myr-(UDCA)2: 3.55nM

[0135] Further results are shown in Figures 7-9.

[0136] In the experiments shown in Figures 7 and 9, the following IC50 values ​​were measured: UDCA: 79μM CA: 115μM MyrB-UDCA: 1.5 nM HBVpreS / 2-21myr (consensus): 70.3nM HBVpreS / 2-21myr-CA: 12.5nM HBVpreS / 2-21myr-UDCA: 9.2nM HBVpreS / 2-21myr-LCA: 7.5nM HBVpreS / 2-21myr-UDCA: 9.2nM HBVpreS / 2-21myr-(UDCA-dimer): 12.7nM

[0137] The IC50 values ​​for the cyclic compounds were all in the range of 11 nM to 100 nM, as shown in FIG.

[0138] [Example 4: Planar imaging] The peptides were radiolabeled with iodine-125 at the tyrosine moiety using the chloramine T method.

[0139] Five microliters of 1 mM peptide / peptide conjugate solution was added to 25 μL of phosphate buffer (0.25 M, pH 7.5). The labeling reaction was initiated by adding 1-20 MBq of iodine-125 in 0.05 M NaOH and 5 μL of freshly prepared aqueous chloramine T solution (2.8 mg / mL). After vortexing for 30 seconds, the labeling reaction was stopped by adding 10 μL of saturated aqueous methionine solution. The radiolabeled peptide / peptide conjugate was purified by semi-preparative radio-HPLC using a Chromolith Performance RP-18e column (100 × 4.6 mm; Merck) eluted with a linear gradient of 0.1% TFA in water and acetonitrile. The solvent of the collected fractions was removed in vacuo, and the labeled product was reconstituted in PBS.

[0140] For in vivo planar scintigraphy imaging of iodine-125-labeled compounds, naive NMRI mice (20–25 g) were anesthetized with 2% sevoflurane. For each compound, 1–5 MBq of radioactivity was injected into the tail vein. Scintigraphy imaging was performed using a Gamma Imager SCT (Biospace Lab, Paris, France) equipped with a parallel collimator (35 mm / 1.8 mm / 0.2 mm). Serial scintigraphy images were scanned at the indicated time points after injection. See Figure 6.

[0141] The features disclosed in the foregoing description, in the claims and / or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

[0142] [References] Blank A, Markert C, Hohmann N, Carls A, Mikus G, Lehr T, Alexandrov A, Haag M, Schwab M, UrbanS, Haefeli WE. First-in-human application of the novel hepatitis B and hepatitis D virus entry inhibitor myrcludex B. J Hepatol. 2016 Sep;65(3):483-9. Bogomolov P, Alexandrov A, Voronkova N, Macievich M, Kokina K, Petrachenkova M, Lehr T, Lempp FA, Wedemeyer H, Haag M, Schwab M, Haefeli WE, Blank A, Urban S. Treatment of chronic hepatitis D with the entry inhibitor myrcludex B: First results of a phase Ib / IIa study. J Hepatol. 2016Sep;65(3):490-8. Chan, HL. & Sung, JJ. Hepatocellular carcinoma and hepatitis B virus. Semin Liver Dis 26, 153-161 (2006). Donkers JM, ZehnderB, van Westen GJP, Kwakkenbos MJ, IJzerman AP, Oude Elferink RPJ, Beuers U, Urban S, van de Graaf SFJ. Reduced hepatitis B and D viral entry using clinically applied drugs as novel inhibitors of the bile acid transporter NTCP. Sci Rep. 2017 Nov 10;7(1):15307. doi: 10.1038 / s41598-017-15338-0. Gripon P, Cannie I, Urban S. Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein. J Virol 2005;79:1613-1622. Lempp FA & Urban A. Hepatitis Delta Virus: Replication Strategy and Upcoming Therapeutic Options for a Neglected Human Pathogen. Viruses. 2017 Jul 4;9(7). pii: E172. doi: 10.3390 / v9070172. Review. Mailly L, Xiao F, Lupberger J, Wilson GK, Aubert P, Duong FH, Calabrese D, Leboeuf C, Fofana I, Thumann C, Bandiera S, Lutgehetmann M, Volz T, Davis C, Harris HJ, Mee CJ, Girardi E, Chane-Woon-Ming B, Ericsson M, Fletcher N, Bartenschlager R, Pessaux P, Vercauteren K, Meuleman P, Villa P, Kaderali L, Pfeffer S, Heim MH, Neunlist M, Zeisel MB, Dandri M, McKeating JA, Robinet E, Baumert TF. Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody. Nat Biotechnol. 2015;33(5):549-54. doi: 10.1038 / nbt.3179. Epub 2015 Mar 23. Gouin-S & Zhu-XX, Synthesis of 3 alpha- and 3 beta-dimers from selected bile acids. Steroids 1996; 61: 664-669. Mentha N, Clement S, Negro F, Alfaiate D. A review on hepatitis D: From virology to new therapies. J Adv Res. 2019 Mar 29;17:3-15. doi: 10.1016 / j.jare.2019.03.009. eCollection 2019 May. Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Falth M, Stindt J, et al. Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes. Gastroenterology 2014;146:1070-1083. Schieck, A., Muller, T., Schulze, A., Haberkorn, U., Urban, S., & Mier, W. (2010). Solid-phase synthesis of the lipopeptide Myr-HBVpreS / 2-78, a hepatitis B virus entry inhibitor. Molecules, 15(7), 4773-4783. doi:10.3390 / molecules15074773 Schulze A, Schieck A, Ni Y, Mier W, Urban S. Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction. J Virol 2010;84:1989-2000. Shepard, C.W., Simard, E.P., Finelli, L., Fiore, A.E. & Bell, B.P. Hepatitis B virus infection: epidemiology and vaccination. Epidemiol Rev 28, 112-125 (2006). Urban S, Bartenschlager R, Kubitz R, Zoulim F. Strategies to Inhibit Entry of HBV and HDV into Hepatocytes. Gastroenterology 2014;147(1):48-64. Wedemeyer H.,…..& Urban S.. 2018. Interim Results of a multicenter, Open-Label Phase 2 Clinical Trial (MYR203) to Assess Safety and Efficacy of Myrcludex B in Combination with Peg-Interferon Alpha 2a in Patients with Chronic HBV / HDV Co-Infection. Hepatology , 68(1):11A. Wedemeyer H., ….& Urban S. 2019. Final Results of a Multicenter, Open-Label Phase 2 Clinical Trial (MYR203) to Assess Safety and Efficacy of Myrcludex B in Combination with Peg-Interferon Alpha 2a in Patients with Chronic HBV / HDV Co-Infection. J.Hepatology, 70(1). Wedemeyer H. ….& Urban S. et al. 2018. Final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with Tenofovir in patients with chronic HBV / HDV co-infection. J Hepatol , 68:S3. Wedemeyer H et al. 2017. Interim results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with Tenofovir in patients with chronic HBV / HDV co-infection. Hepatology, (2017), 66 S1, 20A-21A. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. elife. 2012;1:e00049.

Claims

1. (a) a peptide moiety, and (b) a sodium taurocholate cotransporting polypeptide (NTCP) substrate moiety corresponding to the bile acid binding site of NTCP, the NTCP substrate moiety being a bile acid, a bile acid dimer, or a bile acid multimer. wherein (a) and (b) are covalently bonded to each other; The peptide portion (a) is General formula I: H-[(X) ｍ -P-(Y) ｎ ]-R (I) Hydrophobically modified preS-derived peptides of Hepatitis B virus (HBV) (In the formula, P is the amino acid sequence NPLGFXaaP (SEQ ID NO: 1), where Xaa is F or L; X is an amino acid sequence having a length of m amino acids, where m is 0 or at least 1; Y is an amino acid sequence having a length of n amino acids, where n is 0 or at least 1; where m+n is 5 to 25; H is a hydrophobic modification, located at the N-terminus of P or within X or Y, and selected from acylation with a C8-C22 fatty acid and attachment of a hydrophobic moiety selected from cholesterol, cholesterol derivatives, phospholipids, glycolipids, glycerol esters, steroids, ceramides, and isoprene derivatives; R is a C-terminal modification, a moiety that protects against degradation selected from amides, D-amino acids, modified amino acids, cyclic amino acids, natural polymers, and synthetic polymers; or General formula Ia: Cyclo[(X) ｍ -P-(Y) ｎ ] (Ia) Cyclic peptides (In the formula, P, X, Y, m and n are as defined above; carrying at least one hydrophobic modification in the amino acid side chain of X and / or Y; wherein the cyclic peptide is not cyclized within the amino acid sequence of P in SEQ ID NO: 1 and is not cyclized via the amino acid side chain of P; The hydrophobic modification is acylation with a C8-C22 fatty acid or addition of a hydrophobic moiety selected from cholesterol, cholesterol derivatives, phospholipids, glycolipids, glycerol esters, steroids, ceramides, and isoprene derivatives; or a pharmaceutically acceptable salt thereof A conjugate compound selected from:

2. 2. The conjugate compound of claim 1, wherein the hydrophobic modification of the peptide moiety (a) is acylation with a C8-C22 fatty acid selected from capric acid (C10), lauric acid (C12), myristoyl (C14), palmitoyl (C16) or stearoyl (C18).

3. the peptide moiety (a) is a peptide of general formula I, wherein m=0-18 and / or n=0-7, or 3. The conjugate compound according to claim 1 or 2, wherein said peptide moiety (a) is a cyclic peptide of said general formula Ia, wherein m=0-18 and / or n=0-7.

4. The conjugate compound according to any one of claims 1 to 3, wherein the peptide moiety (a) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 25.

5. the peptide portion (a) comprises a further amino acid for covalently linking the NTCP substrate portion, wherein the further amino acid is an L- or D-amino acid selected from lysine (K), D-lysine (k), D-tyrosine (y), cysteine ​​(C), propargylglycine, azidophenylalanine, azidolysine, azidophenylalanine, homoallylglycine, homopropargylglycine, azidohomoalanine, azidonorleucine, azidophenylalanine, propargyloxyphenylalanine and acetylphenylalanine, and may be a natural or unnatural amino acid; and / or the peptide moiety (a) is a cyclic peptide comprising an additional amino acid for cyclization, wherein the additional amino acid for cyclization can be a natural or unnatural amino acid selected from cysteine ​​(C), allylglycine, propargylglycine, and azidophenylalanine; The conjugate compound according to any one of claims 1 to 4.

6. The NTCP substrate moiety (b) is selected from the following monomeric and polymeric bile acids: Cholic acid (CA); ursodeoxycholic acid (UDCA); Lithocholic acid (LCA); Taurocholic acid (TCA) Glycocholic acid Taurodeoxycholic acid (TDCA) Taurochenodeoxycholic acid (TCDC) and Taurourosodeoxycholic acid (TUDCA) Taurine- or glycine-conjugated bile acids and salts thereof selected from: Sulfated bile acids and their salts; Ursodeoxycholic acid dimer; Dimers containing taurourosodeoxycholic acid (TUDCA); and Mixed dimers of bile acids The conjugate compound according to any one of claims 1 to 5, wherein the compound is selected from the group consisting of:

7. The NTCP substrate portion (b) is UDCA-UDCA, CA-UDCA, and 【Chemical 1】 The conjugate compound of claim 6, wherein the compound is selected from:

8. The conjugate compound according to claim 6 or 7, wherein the NTCP substrate portion (b) is not bound to the peptide portion (a) via the 3-hydroxyl group of ring A of the steroid skeleton.

9. drugs or their respective prodrugs; tag; a label selected from a fluorescent dye, a radioisotope, and an imaging agent; recombinant viruses; a carrier or depot for a drug, prodrug, or label; immunogenic epitopes; hormone; inhibitors; and toxin, 9. The conjugate compound of any one of claims 1 to 8, comprising one or more further moieties selected from:

10. 10. The conjugate compound of claim 9, wherein the further moiety is covalently attached via a linker, spacer and / or anchor group.

11. 11. The conjugate compound of claim 10, wherein the further moiety is covalently attached via a cleavable linker.

12. The conjugate compound according to any one of claims 1 to 11, wherein the peptide moiety (a) and the NTCP substrate moiety are covalently bonded to each other via a linker or an amino acid side chain.

13. (i) at least one conjugate compound according to any one of claims 1 to 12; (ii) optionally, a pharmaceutically acceptable carrier and / or excipient; A pharmaceutical composition comprising:

14. Use of a conjugate compound according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 in the manufacture of a medicament.

15. Use of a conjugate compound according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 in the manufacture of a product for the diagnosis, prevention and / or treatment of liver diseases or conditions.

16. the liver disease or condition is selected from hepatitis, cirrhosis and hemochromatosis; and / or the liver disease or condition is a disease associated with the liver stage of a viral or non-viral pathogen; and / or 16. The use of claim 15, wherein the liver disease or condition is a liver tumor.

17. The liver disease or condition is selected from hepatitis caused by hepatitis A, B, C, D, E, F, G and H viruses or simultaneous hepatitis caused by multiple viruses. and / or the liver disease or condition is selected from tropical diseases, malaria, schistosomiasis, leishmaniasis, Wilson's disease; and / or 17. The use of claim 15 or 16, wherein the liver disease or condition is hepatocellular carcinoma (HCC).

18. the liver disease or condition is a post-transplant complication after liver transplantation related to bile salt accumulation in the bile tract; and / or The liver disease or condition involves sodium taurocholate cotransporting polypeptide (NTCP)-mediated transport of a compound into hepatocytes or requires delivery of a drug or label to the site or location of the disease or condition; and 18. The use according to any one of claims 15 to 17, wherein the compound transported into hepatocytes via NTCP is a bile acid, a steroid, a conjugated and unconjugated thyroid hormone, a hepatotoxin, a compound covalently bound to taurocholic acid, bromosulfophthalein, or a drug.

19. the liver disease or condition is intrahepatic cholestasis, Liver poisoning (due to liver toxins) / hepatotoxicity, Drug-induced cholestatic liver disease, hyperlipidemia, Posthepatic cholestasis, metabolic syndrome, Non-alcoholic fatty liver disease (NAFLD), glycogen storage disease The use according to any one of claims 15 to 18, wherein the liver-related metabolic disease is selected from the group consisting of:

20. Use of a conjugate compound according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 in the preparation of a combination therapy formulation with another therapeutic agent, comprising: The formulation (a) the conjugate compound is formulated to be administered in a therapeutically effective amount in the range of 0.01 mg to 50 mg per patient, or in a dose range of 10 pmol to 20 μmol per kg of body weight to a patient; and / or (b) is designed to be administered by a route selected from oral, subcutaneous, intravenous, nasal, intramuscular, transdermal, inhalation, or by suppository; use.

21. 21. The use of claim 20, wherein the further therapeutic agent is selected from an immunomodulator and / or an agonist or antagonist of RIG-I, MDA-5 or a TLR.

22. The use described in claim 20, wherein the preparation is formulated so that the conjugate compound is administered in a therapeutically effective amount in the range of 0.1 mg to 10 mg per patient.

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